Vaginal laxity therapy utilizing cell-based bulking compositions

ABSTRACT

The invention provides methods for the treatment of vaginal laxity which include delivering a cell-containing composition to the vagina. The composition can include fat tissue to provide a bulking effect to reduce the size of the vaginal opening. The cells can provide healing and revascularization of the vaginal treatment area to sustain the bulking provided by the fat. The invention also provides systems and compositions useful for performing the method, and can include instruments and devices for removal of autologous adipose tissue from a patient (e.g., by liposuction), equipment for the enrichment of cells from adipose tissue, mechanical processing of adipose tissue, and the mixing of cells and processed adipose tissue. Devices for the delivery of the cell compositions to the vagina can also be included in the system.

PRIORITY

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/759,075, filed Jan. 31, 2013, entitled VAGINALLAXITY THERAPY UTILIZING CELL-BASED BULKING COMPOSITIONS, the disclosureof which is incorporated herein by reference.

BACKGROUND

Vaginal laxity is a condition that affects 20 million women in the U.S.alone, ranging in ages 15-49, and is described as a looseness of thevaginal canal or introitus. This condition is most often caused bytrauma from vaginal childbirth delivery and has been identified as alegitimate medical condition by gynecological professionals. Further,most gynecologists identify laxity as the third piece of the triad ofvaginal birth defects that also includes urinary incontinence andvaginal prolapse. Physicians have noted that women who develop vaginallaxity will often later go on to develop vaginal prolapse. Of the 20million women affected by vaginal laxity, approximately half arebothered by it and would like a solution. However, many physicians donot bring it up with their patients because of a lack of treatmentoptions.

SUMMARY

The invention provides systems, methods, and compositions for thetreatment of vaginal laxity that include the use of the patient's ownautologous tissue. The treatment methods of the invention can also beuseful for preventing prolapse, which is thought to follow vaginallaxity later in life if the laxity condition is not treated.

In embodiments, the invention provides a method for treating vaginallaxity. The method comprises a step of providing a compositioncomprising a patient's autologous tissue and/or cells, and also a stepof introducing the composition into the vaginal cavity. The introducedcomposition can bulk the region and reduce the size of the vaginalopening and cavity, thereby treating the vaginal laxity.

The method can optionally include one or more steps to obtain or preparethe composition comprising the patient's autologous tissue and/or cells.For example, a tissue sample can be obtained from the patient andprocessed to isolate or enrich cells for the composition. In some cases,the method includes a step of obtaining an adipose tissue sample fromthe patient, and a step of processing the adipose tissue to isolate orenrich adipose tissue-derived cells, such as adipose tissue-derived stemcells. The method can include steps such as removing the adipose tissuefrom a patient by liposuction, and using tissue processing equipment toisolate or enrich the adipose tissue-derived cells.

The method can optionally include one or more steps of culturing thepatient's autologous tissue and/or cells in vitro to promotedifferentiation towards a desired cellular phenotype, such as vaginalepithelial cells or vaginal muscle cells. Stimulated autologous cells,in a pre-differentiated state, or in a differentiated state, can bedelivered to the vaginal cavity. Optionally, the cells can be deliveredto the vaginal cavity along with a cellular matrix used for cellculturing.

The composition can also include one or more components in addition tothe patient's autologous tissue cells. For example, the composition canfurther include the patient's fat, growth factors, or combinationsthereof. In these embodiments the method can optionally include thesteps of processing adipose tissue sample from the patient (e.g., bymechanical action processing), and then mixing the processed adiposetissue with isolated or enriched adipose tissue-derived cells. In otherembodiments the method further includes mixing one or more growthfactors with the cells, or with a mixture of cells and processed adiposetissue. The growth factors can be obtained from the patient's tissue, orcan be obtained from a different source.

If a composition including processed adipose tissue is used, theprocessed adipose tissue in the mixture can act as the bulking agent,while the cells isolated or can provide healing and revascularization ofthe vaginal treatment area to sustain the bulking provided by theprocessed adipose tissue. Further, the composition can include immunefactors in mixture with adipose-derived stem cells that that can promotedifferentiation of stem cells into muscle cells. Differentiation of thestem cells can lead to strengthening the musculature of the vaginal areaand reduce the risk of, or prevent prolapse later in life.

The invention also provides systems for treating vaginal laxity. In someembodiments the system comprises a composition comprising a patient'sautologous tissue cells, and a device for delivering the composition tothe vaginal cavity. The device can be one that is insertable into thevagina (such as a pessary) that can facilitate desired release of thecells. In other embodiments the system comprises equipment for providinga composition comprising a patient's autologous tissue or cells. Suchequipment can include one or more instruments for obtaining a tissuesample (e.g., adipose tissue) from the patient, such as liposuctioninstruments; a device for enriching cells from the tissue sample; adevice for mechanically processing tissue (e.g., adipose tissue); orcombinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a pessary for delivering a cell composition to thevaginal tissue.

FIG. 2 is a cross-sectional illustration of an article having a doublewalled structure for delivery of cells to vaginal tissue.

DETAILED DESCRIPTION

The embodiments of the present invention described herein are notintended to be exhaustive or to limit the invention to the precise formsdisclosed in the following detailed description. Rather, the embodimentsare chosen and described so that others skilled in the art canappreciate and understand the principles and practices of the presentinvention.

All publications and patents mentioned herein are hereby incorporated byreference. The publications and patents disclosed herein are providedsolely for their disclosure. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate anypublication and/or patent, including any publication and/or patent citedherein.

Women having vaginal laxity (“vaginal looseness”) can have symptoms,issues, and/or experiences such as discomfort and characteristics ofprolapse, tampons falling out of the vagina, lack of friction duringintercourse, penis falling out of vagina during intercourse, vaginalwind, and bathwater entrapment (e.g., see Textbook of Female Urology andUrogynecology, 2nd Edition (2006); Cardozo, L., and Staskin, D., eds.;Informa Healthcare, UK). A healthcare professional, such as agynecologist, can diagnose a female with vaginal laxity based on some orall of these conditions.

Embodiments of the invention include those directed to compositions,systems, and methods for the treatment of vaginal laxity.

Anatomically, the vagina is a muscular orifice having a partiallycollapsed tubular shape, extending from the vestibule of the vulva tothe uterus. The depth of the adult vagina is approximately 4½ to 5inches. The vagina has anterior and posterior walls that are contactwith one another, except at the apex where the vagina surrounds theectocervix. Fornices (vault-like recesses) divide the vagina and cervix.The anterior fornix is shallower than the posterior fornix. Anterior tothe vagina are the urethra and the neck of the bladder. The rectum isposterior to the vagina.

The vagina is lined with stratified squamous epithelium and has crypts(anatomical invaginations into the cellular layer) that produce mucus.Mucus production prevents bacteria from entering the uterus and alsoaids in reproduction when sperm enters the uterus. The superficial layerof vaginal tissue is not keratinized. Microorganisms common to the adultvagina include Doderlein bacilli, diphtheroides, and anaerobicstreptococci. The microorganism flora produces lactic acid from glycogenmade by the vaginal cells. This provides the vagina with a low pH andenhances normal secretions.

Hormones can act upon the vaginal epithelium. Proliferation andmaturation of the epithelium is caused by estrogen action on the tissue,with glycogen accumulating in the epithelial cells.

The term “autologous” refers to material that is obtained from the sameindividual. Autologous tissue and autologous cells are those tissues andcells obtained from, for example, the same patient. Therefore, atreatment that uses autologous cells can be one where an adipose tissuesample is taken from a patient, the patient's autologous cells areobtained from the adipose tissue sample, and then autologous cells arereintroduced into the patient in a desired manner.

In some modes of practice, the composition comprising a patient'sautologous tissue and/or cells is prepared from an adipose tissue samplefrom the patient. In some modes of practice, adipose tissue is removedfrom a desired anatomical location in the patient, processed to providea composition, and then the composition is introduced into the vaginalcavity. Optionally, the composition is cultured before being introducedinto the vaginal cavity. The introduced composition can bulk the regionand reduce the size of the vaginal opening and cavity, thereby treatingthe vaginal laxity. Processed adipose tissue in the composition can actas the bulking agent. In other embodiments a bulking agent that isdifferent than processed adipose tissue can be used. Enriched orisolated cells mixed back in with the processed adipose tissue cansustain the bulking provided by the fat. For example, the isolated cellscan support formation of tissue by providing effects of healing andrevascularization of the vaginal treatment area.

Adipose tissue (i.e., fat) includes or yields a high number of desirablecell types, including stem cells, which can be enriched from the tissueand reintroduced into the patient for revascularization. Adipose tissuecan also be disaggregated and used as a bulking composition.Disaggregated adipose tissue, and cells (e.g., stem cells) enriched orpurified from the adipose tissue can be mixed together and used as thecomposition for introduction into the vaginal cavity. For example, inpreparing the composition, an adipose tissue sample can be obtained andseparated into first and second portions. Cells such as stem cells canbe enriched or purified from the first portion. Adipose tissue from thesecond portion can be disaggregated by mechanical action, such aschopping. Cells enriched from the first portion can then be mixed withdisaggregated adipose tissue from the second portion.

To obtain an adipose tissue sample, a lipectomy surgical procedure canbe performed. As a general matter, during lipectomy fat is removed froman area in the body using a thin metal suction tube introduced through askin incision. The tube is repetitively moved through the tissue tobreak up the fat globules. Vacuum is applied to remove the loosened fatglobules. A medical instrument for performing lipectomy may have ahollow body that can be grasped with the hand. At one end, a suctionprobe is attached to the body and the other end of the body is connectedto a source of vacuum. The probe is used to break the fat globules andthen suction is applied to suck the loosened fat globules out of thebody. The process is repeated until a desired amount of fat is withdrawnfrom the body. Lipectomy methods and instruments are known in the art.For example, see U.S. Pat. Nos. 4,568,332, 5,013,300, and 5,665,101.

Adipose tissue obtained by lipectomy can be processed and the cellpreparation obtained can be mixed with disaggregated adipose tissue andreintroduced into the vagina of the same patient, thereby providing anautologous source of cells.

The adipose tissue can come from anywhere in the body. In oneembodiment, the adipose tissue is obtained from the abdominal area ofthe patient. Other common areas may include the thigh and back area ofthe patient. In some modes of practice, to provide an amount ofcomposition for introduction into the vagina for treatment of vaginallaxity, adipose tissue in an amount in the range of about 50 cc to about200 cc is obtained from the patient. In some modes of practice, theadipose tissue can then be separated into two (or more) portions, onefor preparing the enriched cell population, and the other for preparingthe disaggregated adipose tissue which can serve as the bulking agent aswell as the “cell matrix” for the enriched population of cells.

In some modes of practice, adipose tissue is processed to separate theadipose-derived stem cells from the other material including othercellular and non-cellular material in the adipose tissue. Preparationmethods can include steps of washing the tissue, treating the tissuewith collagenase or trypsin, or optionally with mechanical agitation.Liposomes, which are generally aggregated, can be separated from freestromal cells which include the stem cells and other cells such as redblood cells endothelial cells, and fibroblast cells, by centrifugation.Erythrocytes can be lysed from the suspended pellet and the remainingcells can be filtered or centrifuged. Optionally, cells may be separatedby cell sorting or separated immunohistochemically. Methods for thepreparation of adipose-derived stem cells are described incommonly-assigned application number WO 2009/120879.

In other modes of practice, the adipose tissue is processed to removepartially or substantially non-cellular components, and to form aheterogenous cell mixture. The heterogenous cell mixture can includeendothelial cells, endothelial precursors and progenitors, mesenchymalstem cells, vascular smooth muscle cells, fibroblasts, pericytes,macrophages, and the like.

PCT Application PCT/US2010/041508 describes methods and apparatus forthe preparation of cellular material. The methods and apparatus can beused in the method and systems of the current invention. Cell separationequipment is also commercially available from, for example, TissueGenesis, Inc. (Honolulu, Hi.).

In some modes of practice tissue derived (e.g., adipose-derived)autologous stem cells can be treated to induce their differentiationtowards a desired cell type. After obtaining from a patient, the sampleof autologous cells including stem cells can be placed in a cell cultureenvironment in contact with a cell differentiation factor for a periodof time. The period of time can be minutes, hours, days, or weeks,depending on desired stimulatory parameters for cell differentiation.The cells can be placed in a desired cell culture container to promotedifferentiation and optionally expansion. Examples of cell culturecontainers include flasks, dishes, plates, and multi-well plates.

In this case, the liposuction procedure, cell enrichment, cellculturing, and reintroduction into a patient may be performed in asingle clinic or hospital visit. Culturing in this mode of practice maybe carried out over a short period of time, such as on the order ofminutes of hours. In other cases, cell culturing may be performed overthe course of a day or days, or weeks. In these scenarios, the patientmay have two or more office clinic or hospital visit. For example, afirst visit to perform the liposuction procedure and begin the cellenrichment and cell culturing procedures, and a second visit tointroduce the cultured cells into the patient.

In vitro culturing can include use of cell scaffolds to promotedifferentiation and growth of the cells. Exemplary scaffolds includedegradable materials and non-degradable materials. Exemplarynon-degradable (which otherwise may be referred to as biostable,non-absorbable) materials include, but are not limited topoly(vinylchloride), polystyrene, poly(vinylidene fluoride),poly(vinylidene chloride), polymethylmethacrylate (or other acrylicresins), polyamide, polyester, polyarylene oxide, polysulfone,polyethylene, polypropylene, and polyvinyl alcohol. Cultured cells canbe separated from the non-degradable materials prior to reintroductionin the body.

Exemplary degradable (e.g., bioabsorbable) materials include degradablealiphatic polyesters such as polylactic acid,poly(lactide-co-glycolide), polycaprolactone, poly(valerolactone),poly(glycolic acid), poly(glycolide), and poly(dioxanone);polyhydroxyalkanoates, such as poly-4-hydroxybutyrate (P4HB),poly(3-hydroxyvalerate, and poly(hydroxybutyrate-co-hydroxyvalerate);polyorthoesters; polyalkeneanhydrides, such as poly(sebacic acid);polyanhydrides, polyphosphazine; and natural degradable polymers such ashyaluronic acid, alginate, dextran, cellulose, xanthan, pullulan,chitosan, pectin, and inulin. Cultured cells can be separated from thedegradable materials prior to reintroduction in the body, or can be leftin contact with the degradable materials as they will be absorbed by thebody after a period of time following cell delivery.

The cells can be cultured on a scaffold that includes one or moreextracellular matrix (ECM) component(s). ECM components include fibrousproteins such as collagen; elastic proteins such as elastin;glycoproteins such as fibronectin; and binding proteins such as laminin;glycosaminoglycans heparin sulfate, chondroitin sulfate, keratansulfate, and hyaluronic acid. Cellular scaffolds including ECMcomponents are commercially available (e.g., BD Matrigel™ BasementMembrane Matrix, which is a solubilized basement membrane preparationincluding laminin, collagen IV, heparan sulfate proteoglycans, andentactin/nidogen. Matrigel™ also contains TGF-beta, epidermal growthfactor, insulin-like growth factor, fibroblast growth factor, tissueplasminogen activator, 3,4 and other growth factors which occurnaturally in the EHS tumor.)

If a scaffold is used to culture cells it can include the polymermaterial (e.g., either biostable or biodegradable) in a fine fibrousform. Fine fibers of polymeric material for a cell scaffold can beprepared using an electrospinning process. Electrospinning uses anelectric field to during the formation of the polymeric fibers.Exemplary electrospinning processes are described in U.S. Pat. No.6,743,273 and U.S. Publication No. 2006/0290031.

Fine fiber scaffolds can have directed or random orientations, andoptionally be layered. The fine fiber scaffolds can have very smallspaces (e.g., pores) between the fibers that can permit the penetrationand in-growth of cells and that also ready permit diffusion of cellculture materials such as liquids, ions, proteins, and active factors inand out of the scaffold.

Other fine fiber processing techniques include polymer extrusion. Anexemplary fine fiber polymer scaffold is formed from extrudedpolyglycolic acid (PGA). For example, Freed L. E., et al. (Biotechnology(N.Y) 199412:689-93) describes the extrusion and orientation ofmicron-sized PGA fibers. Textile processing techniques can be used toproduce fibrous scaffolds with a high porosity percentage and goodstructural integrity. Degradable fibers can optionally be introducedinto the body along with the autologous cells, as the fibers willeventually be broken down and absorbed by the body. The fibers can alsoinduce cell differentiation with respect to morphology and phenotype.

In some modes of practice, the cells seeded on a biodegradable scaffold,and then the cell scaffold is placed within the vaginal cavity of thepatient in need of treatment. The cell scaffold can be cultured for adesired period of time prior to placing in the patient. The cellscaffold can also be configured for placement within the confines of thevaginal cavity. For example, the scaffold can be cut to provide adesired shape of material appropriate for contact with a vaginal wall ofthe cavity.

The cell scaffold can also be associated with an article configured forinsertion into the vagina, such as a pessary, tampon, vaginal ring,suppository, or vaginal sponge. For example, the cell scaffold is in theform of a layer on the article (e.g., pessary) surface. The article mayhave multiple layers of biodegradable cell scaffolding on its surface.In use, the biodegradable scaffolding can degrade over a period of timeand transfer the cells therein to the vaginal tissue in need oftreatment. Exemplary pessaries are described in U.S. Pat. Nos.3,554,184, 5,224,494, 6,158,435, 6,428,467, and 6,645,137.

As used herein, a “differentiation factor” refers to a molecule havingbioactivity capable of causing cellular differentiation. Celldifferentiation refers to a process of a generic cell (e.g.,non-specific) developing into one that is more specific. For example, adifferentiation factor can act on a totipotent cell or pluripotent cellto develop the cell into a certain cell type. Proliferation of the cellcan also occur during cell differentiation. The term “growth factor” asused herein refers a molecule having bioactivity capable of causing thecell or tissue proliferation. A growth factor can act on anundifferentiated cell or a differentiated cell to cause theirproliferation. In many cases the growth factor can also cause celldifferentiation.

Exemplary growth and/or differentiation factors include transforminggrowth factor-alpha (TGF-α); transforming growth factor-beta (TGF-β),including TGF-β1, TGF-β2, and TGF-β3; colony stimulating factor (CSF),such as granulocyte-CSF, macrophage-CSF, granulocyte-macrophage-CSF;platelet-derived growth factors (PDGF) such as PDGF-AA, PDGF-AB andPDGF-BB isoforms; nerve growth factor (NGF) such as NGF 2.5s, NGF 7.0s,and β3-NGF; neurotrophins and brain derived neurotrophic factor;fibroblast growth factor (FGF) such as FGF acidic isoforms 1 and 2, FGFbasic form 2, and FGF 4, 8, 9 and 10; vascular endothelial growth factor(VEGF), EG-VEGF, VEGF-related protein, Bv8, and VEGF-E; insulin likegrowth factor (IGF) I and II; cartilage derived factor; bone growthfactor (BGF); erythropoietin; IL-3; hepatocyte growth factor; glialneurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocytegrowth factor (KGF); skeletal growth factor; bone matrix derived growthfactors, and bone derived growth factors and mixtures thereof.

In some mode of practice, the cells can be cultured with one or moregrowth and/or differentiation factor(s) prior to delivering thestimulated cells to the vaginal cavity. In other modes of practice, thecells are contacted with the one or more growth and/or differentiationfactor(s) while the cells are being delivered to the vaginal cavity, orafter the cells have been delivered to the vaginal cavity.

During in vitro cell culturing, or in vivo seeding, the composition caninclude use of one or more cell attachment factors. Cell attachment anddifferentiation to a desired phenotype can be promoted by using an ECMfactor such as collagen, fibronectin, laminin, or a glycosaminoglycan(e.g., keratin sulfate, heparan sulfate, chondroitin, dermatan sulfate).Peptides with active motifs for cell attachment can also be used. Othercomponents useful for cell attachment include agar, agarose, gelatin,gum arabic. Cell attachment factors can be associated with a cellscaffold for in vitro culturing. In another mode of practice the cellattachment factor is provided to the vaginal cavity before or duringintroduction (or both) of the autologous cells in order to promote cellattachment to the existing vaginal tissue of the patient.

Stem cells that have been induced to differentiate towards a vaginalcell type, such as vaginal epithelial cells or vaginal muscle cells canoptionally be monitored in vitro or in vivo to assess differentiationtowards the desired phenotype.

The term “vaginal epithelial cells” are those cells that havecharacteristics of epithelial cells native to the female vagina. Vaginalepithelial cells include cell types of parabasal epithelial cells,intermediate epithelial cells, and superficial cells epithelial cells.Parabasal cells are the smallest epithelial cells, have a round shape,and a high nuclear to cytoplasmic ratio. Intermediate cells typicallyhave a diameter two to three times that of parabasal cells. Intermediatecells can be subclassified into small intermediate cells, having roundor oval shapes and large distinct nuclei, and large intermediate cellshaving polygonal shape with a low nuclear to cytoplasmic ratio.Superficial cells are the largest vaginal epithelial cells, arepolygonal in shape and noticeably flat, and do not have an observablenuclei or have a nuclei that is pyknotic (very small and dark). The term“fully cornified” refers to superficial cells lacking nuclei. Somevaginal epithelial cells can also be desribed as “squamous,” as presentin squamous epithelium which is characterized by a superficial layerwherein the cells are flat, scale-like. Vaginal epithelial cells canalso include endometrial cells.

Vaginal epithelial cells can also be characterized by the presence ofcertain biomolecular marker on the surface of the cells, or within thecells. Stem cells may differentiate towards a vaginal epithelial celltype that present a cytokeratin marker. For example, the epithelialcells can be examined for the presence of cytokeratin intermediatefilaments (e.g., see, Classen-Linke et al. (1997) Cell Tissue Res.287:171-185) which is often used for characterization. Vaginalepithelial cells can be identified or characterized using monoclonalanti-pancytokeratins AE1/AE3, which bind to many acidic and basiccytokeratin proteins. Cytokeratins are intermediate filament proteinspart of the cytoskeletal componentry.

The AE3 monoclonal antibody recognizes the 65 to 67 triplet, 64, 59, 58,56, and 52 kD proteins also known as cytokeratin 1, 2, 3, 4, 5, 6, 7,while the AE1 antibody recognizes 56.5, 50, 50′, 48, and 40 kDa proteins(also known as CK10, 14, 15, 16 and 19). Anti-cytokeratin antibodies arecommercially available from, for example, eBioscience, Inc. (San Diego,Calif.).

Vaginal epithelial cells also have a relatively high intracellularglycogen concentration. Lugol staining, which uses iodine and potassiumiodide, can be used for detection of vaginal epithelial cells inpreviously existing or newly formed vaginal tissue (e.g., see, Cibas andDucatman (2009) Cytology; Chapter 1: Cervical and Vaginal Cytology,Saunders). Other epithelial markers include progesterone receptors, andestrogen receptors (Cooke et al. (1986) Proc. Natl. Acad. Sci. USA83:2109-2113).

Functionally, vaginal epithelial cells include receptors, signaltransduction mechanisms, and cytokine secretion capabilities forpromoting host defenses against various microorganism infections.

Following a period of in vitro culturing, or a period of in vivoimplantation, adipose derived stem cells that have been promoted todifferentiate into vaginal epithelial cells can be tested for thepresence of one or more vaginal epithelial cell markers. Vaginalepithelial cells differentiated from adipose stem cells can be describedin terms of one or more of these markers.

Vaginal smooth muscle cells can be detected using various techniques,such as by immunostaining with antibodies against alpha smooth muscleactin. Smooth muscle cells can also have prostaglandin E andbeta-adrenergic receptors which can trigger increased intracellular cAMPsynthesis in response to PGE1 (or isoproterenol). Smooth muscle cellscan also display enhanced intracellular cGMP synthesis and accumulationwhen treated with a nitric oxide donor in the presence of a PDE type 5inhibitor.

Vaginal epithelial or muscle cells can be characterized using cellspecific antibodies, such as anti-cytokeratin or anti-smooth muscleα-actin antibodies. In some forms, Western blot analysis can be used todetect protein expression in a cell sample (e.g., from either in vitroculture or from a sample taken from the body) to confirm differentiationinto epithelial and smooth muscle cell lines. Tissue sample explantedfrom the patient following treatment can also be examined for thepresence of epithelial cells and/or muscle cells from the newly-formedvaginal tissue. In some cases, formalin-fixed paraffin-embedded tissuescan be prepared and stained with antibodies to determine the presence ofepithelial cells and/or muscle cells.

During in vitro cell culturing, or in vivo seeding, autologous stemcells can differentiate to cells suitable for forming female vaginaltissue having structures and morphology of native vaginal tissue. Theformed tissue can have a physiology that resembles that of normal femalereproductive tissue, such as being responsive to hormones and sensorystimulation similar to a normal vagina. Estrogen stimulates theproliferation and maturation of the vaginal epithelium whileprogestogens inhibit maturation of the epithelium. As another functionalproperty, vaginal cells derived from the stem cells can be capable ofproducing mucus.

Optionally, newly formed vaginal tissue can be tested for its ability toproduce contractile forces (like native vaginal tissue) followingelectrical stimulation. Following stimulation, functioning vaginaltissue can be characterized by cell depolarization and the release ofintracellular cations resulting in a contractile force. Othercharacteristics include a response to stimulation using a muscarinic oran adrenergic agonists, suggesting the presence of neurotransmitterfunction.

The cell composition delivered to the vaginal cavity can also promoteangiogenesis or neovascularization. Angiogenesis is a process of newblood vessel development and plays an important role in the femalereproductive cycle e.g., ovulation, menstruation and placentaldevelopment. The endothelial cell population can be used to stimulatevascularization. The cells can be treated with a factor that promotesangiogenesis, such as VEGF-C, which is implicated in angiogenesis.(Yancopoulos, G. D., et al. (2000) Nature, 407:242-248. After a periodof treatment, newly formed vaginal tissue can optionally be analyzed forthe formation of new blood vessels.

In some modes of practice after a population of the adipose-derivedcells (e.g., stem cells; ADSCs) is enriched, the cells can be introducedinto the vagina. As discussed herein, the stem cells can optionally becultured prior to introducing into the vaginal cavity.

In other modes of practice, the adipose-derived cells are mixed with thedisaggregated adipose tissue to form a composition which is thenintroduced into the vagina. The adipose tissue can be disaggregated bymechanical force, such as by cutting, chopping, or mincing the adiposetissue. Generally, during preparation using mechanical action,collagenase or trypsin (enzymatic) digestion is not performed tomaintain scaffolding and structured features of the adipose tissue.Adipose particles generated using such a process can be sized fordelivery into the vagina. Grinding and filtering parameters can also beemployed depending on the particular treatment site needs.

In some preparations, the isolated or enriched cells are mixed with thedisaggregated adipose tissue at a weight ratio in the range of about 1:1to about 1:4 (cells: tissue). Methods for the preparation of an adiposetissue-derived scaffolding for cells are described in commonly assignedInternational Application PCT/US2009/038426 (WO2009/120879), and aredescribed further herein with reference to the multi-chamber mixing anddelivery systems.

Systems and methods of the invention can also include devices, tools,and methods for the preparation of a composition containing a cellpopulation derived from adipose tissue.

The cell containing composition for introduction into the vagina can beof a desired volume based on factors such as the size of the treatmentarea, and the method and apparatus used to deliver the composition. Asingle injection can be used to deliver the composition to the vagina,or introduction of the composition can occur over multiple injections orintroductions.

The cell-containing composition can optionally include biologics ordrugs which can enhance the effectiveness of the cells followinginjection into the vagina, or that can further improve the condition ofthe tissue. Optionally, the cell-containing composition can includeexcipients, additives, or auxiliary substances useful in the vagina.

Multi-chamber mixing and delivery systems can be used to preparecell-containing compositions, such as ADSC compositions, forintroduction into vagina. The devices can also be used for the injectionprocess. Mixing and delivery systems can include various components andelements to facilitate mixing, digesting, filtering and introduction ofcellular mixtures, e.g., cells and autologous adipose tissue orscaffolding material, into the vagina for treatment of vaginal laxity.The various components of the systems can be constructed of materialssuch as polymers, metals, and other like materials compatible for usewith such injection systems and methods.

Devices, methods, and compositions prepared therefrom, including thosedisclosed in U.S. Patent Publication Nos. 2005/0177100, 2006/0100590,2007/0224173, 2008/0014181, 2008/0287879 and 2009/0018496; U.S. Pat. No.7,101,354; and PCT International Patent Publication No. WO2008/091251can be used are envisioned for use in the systems and methods of thecurrent invention, and their disclosures are incorporated herein byreference in their entirety.

The system of the invention can include an injection device, andoptionally one or more components for the removal of adipose tissue, theenrichment of adipose derived stem cells, and/or the mixing of enrichedor isolated adipose stem cells with a mechanically processed adiposetissue. Other system components which can optionally be incorporated inoptional steps of the invention include anesthetics and antibiotics;surgical instruments such as scalpels, forceps, needles, and sutures;and bandages and tapes. The optional components can be used to numb,prevent infection, and/or repair tissue in the patient. Tissue sitesaffected by treatment or surgical intervention include the vagina and atissue site from which adipose tissue can be removed from the patient,such as the abdomen.

Introduction of the composition into the vagina can be carried out usingany suitable delivery conduit, such as a needle, trocar, cannula,catheter, or other tube or hollowed cylindrical article. For example, asyringe and needle combination can be used to control the flow of thecomposition manually (i.e., with forces from the hand) by a practitionerduring administration.

In some modes of practice, the composition is injected into the vaginausing high pressure injection. Use of an injection device that includesa pump to generate the high pressure is one technique for carrying outthe collagenase injection. In some arrangements, the injection devicecan include multiple ports or multiple needles to push the compositionout at a fast rate into the vagina. The process of high pressureinjection may be referred to as “jet injection.” Device embodiments forintroducing the composition can include multiple apertures or multipleneedles such as an extended, expanded, or extendable chain, string,array, or sequence (e.g., “daisy chain”). Apertures may be located at anextension mechanism (“aperture extension”) such as extendable or fanningneedles.

The pressurized fluid source can include a source of fluid and a sourceof pressure. The pressure source can include a mechanical feature (suchas a spring), a pneumatic feature, a hydraulic feature, or an electricfeature, etc., to generate the fluid pressure. Further, the pressuresource may be mechanically or electronically controlled. The pressuresource can cause a fluid contained in a fixed or variable volume chamberto be pressurized to a transient pressure, at the injection orifice,that is sufficiently high to allow the fluid to be ejected from theinjection orifice with sufficient force to penetrate the vaginal tissue.

Various device structures, components, methods and techniques describedand depicted in U.S. Patent Publication No. 2006/0129125 andInternational Publication No. WO2007/079152 are envisioned for use,alone or in combination, with the present invention.

The composition can optionally be delivered through an articleconfigured for insertion into the vagina, such as a pessary, balloondevice, or vaginal ring capable of receiving and distributing the cellcontaining composition in the vagina. The article can be of any shapeand size suitable for insertion into the vagina and delivery of

For example, the article can receive the cell composition from anexternal source, and then distribute the composition intravaginally. Thedistribution of the cell composition can occur by the composition movingfrom an internal portion of the article, through one or more aperturesor openings in an outer material of the article, and out to the vaginaltissue where it is desired for the cells of the composition to seed andgenerate new vaginal tissue.

In one arrangement, the composition is introduced into the vagina usinga balloon-based injection system. For example, a balloon is inflated inthe vagina or the site of laxity. The balloon contains fluid pathwaysand/or micro-needles along the surface to provide a quick andhomogeneous injection into the vaginal wall. The balloon can be attachedto a distal end of a catheter tube that is in fluid communication withthe inflation lumen. The proximal end of the catheter tube can havefeatures for controlling movement of the catheter and balloon duringinsertion, and controlling inflation of the balloon.

In a similar arrangement, the article can be constructed to have aninner space capable of receiving the cell composition, and a wall ormembrane that can contact a vaginal wall, and having openings throughwhich the cell composition can flow through. FIG. 1 shows a pessary 10that is capable of delivering a cell composition to the vaginal tissuehaving a plurality of openings 12 in its outer wall. Preferably thematerial of the outer wall is pliable to it can change in shape and/orsize, such as during insertion into the vagina, or removal therefrom,during delivery and release of the cell composition, and also aftergrowth of tissue in the vagina. The outer wall can be constructed from abiocompatible and non-absorbable material, such as silicone rubber,nylon, polytetrafluoroethylene (PTFE), a polyamide resin, polyesters, ororganosiloxanes.

The apertures in the wall of the article can be sized to allow thepassage of cells from the inner space out to the vaginal tissue. Forexample, the apertures can have a diameter of at least 25 μm, or morepreferably, greater than 50 μm, or greater than 100 μm, such as in therange of about 100 μm to about 1 mm or 2 mm. The apertures may increasein size upon expansion of the article.

The article 10 can have a portion with a hub 14 connected to a distalend of a tube or conduit 16 that can supply the inner space of thedeliver article with the cell composition. A proximal end of the conduit(not shown) can be attached to a reservoir that supplies the cellcomposition. The reservoir can also provide a source of pressure causingpressurization of the inner space and movement of the cell compositionout through the pores.

In one arrangement, with reference to FIG. 2, the article 20 can have adouble walled structure. FIG. 2 shows a cross-sectional portion of thearticle, with the outer wall 24 having apertures 26, and an inner wall30 without apertures. An inner space 34 is defined between outer 24 andinner 30 walls through which a cell-containing composition can flow. Fordelivery, cells 28 can move through apertures 26 and to the vaginaltissue 22 in which they seed and expand to form new tissue Inner space32 represents an area that can be inflated with a liquid or a gas toprovide pressure to expand the outer wall to the vaginal tissue and/orcause the flow of the cell containing composition out of the apertures26.

After delivery of a desired amount of cell composition, the article canbe removed from the vagina, or maintained in the vagina. If the articleis maintained, the process of cell delivery can be repeated one or moretimes to provide additional cell composition to the vaginal walls for anincreased bulking effect.

Treatment can optionally be carried out using a drug that provides abeneficial therapeutic effect to the vaginal tissue. The drug may beused to enhance the seeding of the autologous cells to the vaginalcavity, to affect newly formed vaginal tissue as a result of the cellseeding, or to provide preventative treatment in order to minimize oreliminate the adverse biological conditions (e.g., such as infection ornecrosis). The drug can be orally or locally (i.e., directly into thevaginal cavity) delivered.

In some modes of practice, a steroid drug is administered to the patientbefore delivery of the cell composition, during delivery of the cellcomposition, after delivery of the cell composition, or combinationsthereof. Exemplary steroid drugs include, but are not limited to anestrogen, such as ethinyl estradiol, 17-estradiol, diethyl stilbesterol,estradiol 3-benzoate, estradiol 17-benzoate, estradiol17-cyclopentylpropionate, estradiol dipropionate, and ethinyl estradiol.In some modes of practice an anti-inflammatory drug is delivered before,during, after, or combinations thereof. Exemplary anti-inflammatoriesinclude triamcinolone, prednisolone, hydrocortisone, cortisone,dexamethasone, and fludinolone. In some modes of practice an antibioticor antimicrobial is delivered before, during, after, or combinationsthereof. Exemplary antibiotics and antimicrobials include erythromycin,penicillin, tetracycline, miconazole, bacitracin, and nystatin. In somemodes of practice a progestogen is delivered before, during, after, orcombinations thereof. Exemplary progestogens include progesterone,norethynodrel, norethindrone, megestrol, melengestrol,medroxyprogesterone, 19-norprogesterone, and 17-hydroxyprogesterone. Insome modes of practice an anti-inflammatory prostaglandins, such asPGE₁; PGE₂, and PGF₂, is delivered before, during, after, orcombinations thereof.

What is claimed is:
 1. A method for treating vaginal laxity, comprisingsteps of: providing a composition comprising autologous tissue, cells,or both, from adipose tissue of a patient; introducing, by aballoon-based injection system, the composition into a vaginal cavity totreat the vaginal laxity, the balloon-based injection system including apessary, the pessary including an inner wall, an outer wall, and a spacedisposed between the inner wall and the outer wall, the outer walldefining a plurality of apertures, wherein the introducing includesinflating the space of the pessary with the composition to expand theouter wall to vaginal tissue causing a flow of the composition out ofthe plurality of apertures; and reducing a size of the vaginal cavity inresponse to the introduction of the composition into the vaginal cavity.2. The method of claim 1, wherein the step of providing comprisesobtaining a tissue sample from the patient and processing the sample toisolate or enrich the cells for the composition.
 3. The method of claim1, wherein the step of providing comprises removing the adipose tissuefrom the patient by liposuction, and using tissue processing equipmentto isolate or enrich adipose tissue-derived cells from the adiposetissue.
 4. The method of claim 1, wherein, in the step of providing, thecomposition comprises, in addition to autologous cells from adiposetissue, adipose tissue processed by mechanical action.
 5. The method ofclaim 4, wherein the adipose tissue processed by mechanical action actsas a bulking agent, and the autologous cells promote healing andrevascularization of tissue in the vaginal cavity.
 6. The method ofclaim 1, wherein the step of providing, the composition comprises growthfactors.
 7. The method of claim 1, wherein the composition comprisesimmune factors in mixture with adipose-derived stem cells that promotedifferentiation of adipose-derived stem cells into muscle cells.
 8. Themethod of claim 7, wherein differentiation of the stem cells into musclecells provides strengthening the musculature of the vaginal cavity andreduces the risk of, or prevents, vaginal prolapse.
 9. The method ofclaim 1, wherein an amount of the adipose tissue is in the range of 50cc to 200 cc.
 10. A method for treating vaginal laxity, comprising stepsof: providing a composition comprising autologous tissue, cells, orboth, from adipose tissue of a patient; placing the compositioncomprising autologous tissue, cells, or both, on a biodegradablescaffold to form a cell-containing biodegradable scaffold; disposing thecell-containing biodegradable scaffold on a surface of a pessary;introducing the pessary having the cell-containing biodegradablescaffold into a vaginal cavity to treat the vaginal laxity; and reducinga size of the vaginal cavity in response to the introduction of thepessary having the cell-containing biodegradable scaffold.
 11. Themethod of claim 10, wherein the cell-containing biodegradable scaffoldis configured for placement within the confines of the vaginal cavity.12. The method of claim 10, wherein the cell-containing biodegradablescaffold comprises a fine fiber polymer scaffold that is formed fromextruded polyglycolic acid (PGA).
 13. The method of claim 10, whereinthe cell-containing biodegradable scaffold comprises a syntheticbiodegradable polymer.